The overriding goal of this proposal is the elucidation of the mechanism of biologic calcification. It is hypothesized that the deposition of mineral on the collagen of bones and teeth is regulated by extracellular matrix macromolecules, with collagen providing an oriented template for mineral deposition, and matrix proteins associated with the collagen or other structures serving as mineral nucleators and regulators of the size and shape of mineral crystals. The capacity of a matrix component to act as a nucleator or mineral growth and proliferation regulator is dependent on the way it interacts with mineral nuclei and crystals once they begin to form. There is extensive data indicating that phosphorylated matrix proteins are important regulators of biomineralization. The gamma-carboxyglutamlc acid containing proteins, which may also be phosphorylated, appear more important for the regulation of crystal size and mineral turnover (remodeling). The mechanism by which these families of proteins perform these functions has not been determined. The aim of this study is to identify the mechanism of matrix mediated calcification of bone and dentin with specific emphasis on the roles of phosphoproteins and gamma-carboxylated proteins. The phosphoproteins will be studied in the gelatin-gel apatite formation system, by Fourier Transform (FT) infrared (IR) analysis of protein conformation, and by chemical or synthetic modification of their structure. Verification of their functions will be provided by FT-IR microspectroscopy of the bones (and teeth) of diseased, transgenic and mutant animals, including osteocalcin, matrix gla protein, and bone sialoprotein "knockout" mice. Five specific hypotheses will be tested: 1) Mineralization of teeth and bones is dependent on the presence of one or more extracellular matrix phosphoproteins which can both promote and regulate apatite formation and proliferation in vitro, and, most likely, in vivo; 2) The effects of these proteins on mineralization are due to the interactions of anionic moieties with the mineral and can be modified by altering the extent of phosphorylation and/or other aspects of the proteins' structure; 3) There are structural domains within these proteins which, when altered, will affect the protein's ability to affect mineralization; 4) These affects are attributable to the conformation the protein has in the mineralizing/mineralized matrix; and 5) The significance of the protein can be demonstrated by analyses of the mineral formed in the bones and teeth of transgenic and knockout animals.